Focusing agents and methods of using same

ABSTRACT

A method for reducing the variability, as measured by relative standard deviation (RSD), of an analytical testing technique is provided. This improvement in RSD improves the confidence in the values obtained during field testing. The method includes incorporating a focusing agent into the sampling media, which permits providing sampling media such as thermal desorption tubes preloaded with the focusing agent.

RELATED APPLICATION

The present application is a continuation application of co-pending U.S.application Ser. No. 15/451,438, filed Mar. 7, 2017, which claimspriority to U.S. Provisional Patent Application Ser. No. 62/305,395filed Mar. 8, 2016. The disclosure of each of these applications ishereby incorporated herein by reference in its entirety.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

FIELD OF THE INVENTION

The invention is related to improved analytical techniques useful forthe identification and enhanced quantification of chemical species, suchas chemical warfare agents.

BACKGROUND OF THE INVENTION

The detection and quantification of chemical warfare agents (CWAs) andtoxic industrial chemicals/materials (TIC/TIM) require field analysisreliance on previously established calibration curves programmed onfield instruments. Sampling tubes constructed with adsorbent materials(which are better known as thermal desorption tubes (TD)) are currentlyemployed for field investigations, and the measured values of the CWAs,TICs, and TIMs demonstrate wide variability when control studies measurerelative standard deviation (RSD).

Accordingly, disconnects exist between current field collection ofsamples and final analysis, and yield data that has high variability andtherefor lower reliability. For example, current studies employing anexemplary portable gas chromatograph-mass spectrometer (GC/MS) HAPSITE®ER (Inficon GMBH) demonstrate wide variability of data. This variabilityis observed both for intra-instrumental and inter-instrumental analyses.Current experiments have demonstrated relative standard deviations (RSD)of about 70%, where the industry standard is generally about 30% RSD orless. This elevated uncertainty is especially surprising consideringthat the device houses a canister of an internal standard (i.e.,bromopentafluorobenzene) that is capable of being injected on eachsample analysis. Similar variability has been observed with comparabledevices from other manufacturers.

Accordingly, there is a need for new testing protocols and equipment toenhance the reliability of these field measurements.

SUMMARY OF THE INVENTION

The present invention is premised on the realization that incorporationof an internal standard (hereinafter referred to as a “focusing agent”)into an analytical sampling device by embedding a known quantity of thefocusing agent onto a media component of the analytical sampling devicecan be leveraged to improve and enhance the reliability of analyticaltesting methods for quantifying chemical compound(s) of interest. Moreparticularly, the present invention is premised on the realization thatrelative retention time (RRT) ratios and relative response factor (RRF)between the focusing agent and the chemical compound(s) of interest maybe correlated to improve a relative standard deviation in the analyticaltesting method.

In accordance with an embodiment of the present invention, a system foridentifying and quantifying a chemical agent is provided. The systemcomprises an analytical sampling device operably connected to achromatographic subsystem and a mass spectrometer subsystem. Theanalytical sampling device comprises an adsorbent material, and a knownquantity of a focusing agent adsorbed therein, wherein the focusingagent is characterized as having a chromatographic or spectralsimilarity to the chemical agent being quantified.

In accordance with another embodiment of the present invention, a methodfor reducing a relative standard deviation (RSD) in an analyticalchromatographical testing procedure for quantifying a chemical agent isprovided. The method comprises obtaining an analysis sample with ananalytical sampling device to pre-concentrate the chemical agent from afluid test sample. The analytical sampling device comprises an adsorbentmaterial, and a known quantity of a focusing agent adsorbed therein,wherein the focusing agent is characterized as having a chromatographicor spectral similarity to the chemical agent being quantified. Themethod further comprises desorbing the focusing agent and the chemicalagent from the analytical sampling device to form a desorbed mixture andtransferring the desorbed mixture into a chromatographic subsystem and amass spectrometer subsystem; applying the analytical chromatographicaltesting procedure to the test sample to obtain a first signal relatingto the focusing agent and a second signal relating to the chemicalagent; and correcting the first signal relating to the focusing agentand the second signal relating to the chemical agent based on apredetermined relative retention time ratio and a relative responsefactor between same.

The objects and advantages of the present invention will be furtherappreciated in light of the following detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with a general description of the invention given above, andthe detailed description given below, serve to explain the invention. Itwill be appreciated that for purposes of clarity and where deemedappropriate, reference numerals have been repeated in the figures toindicate corresponding features.

FIG. 1 is a simplified schematic of a system for identifying andquantifying a chemical agent, in accordance with an embodiment of thepresent invention;

FIG. 2 is cross-sectional view of an analytical sampling device takenalong line A-A of the system shown in FIG. 1;

FIG. 3 is a three-dimensional plot of data showing a three-point curveof an analytical sampling device embedded with a focusing agentcomprising three C-13 isotope analogs of diethyl malonate, where thedata was obtained with a system and method in accordance withembodiments of the present invention;

FIG. 4 is a three-dimensional plot of data of a three-point curve of ananalytical sampling device applied with test sample of diethyl malonate,where the analytical sampling devices was previously embedded with afocusing agent comprising three C-13 isotope analogs of diethylmalonate, where the data was obtained with a system and method inaccordance with embodiments of the present invention;

FIG. 5 is a linear plot of the focusing agent comprising three C-13isotope analogs of diethyl malonate, where analysis was performed onthree instruments over three months in triplicate to demonstrate thelinearity, reproducibility, and stability of the system of theembodiments of the present invention;

FIG. 6 is a linear plot showing the normalized data shown in FIG. 5; and

FIG. 7 is line plot showing percent recoveries of the embedded focusingagents comprising three C-13 isotope analogs of diethyl malonate.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1, a simplified schematic of a system 10 foridentifying and quantifying a chemical agent is shown. The system 10includes an inlet 12 fluidly coupled to an analytical sampling device 14operably connected to chromatographic subsystem 16 and a massspectrometer subsystem 18. Optionally, a pre-concentrator 20 may bepositioned in between the analytical sampling device 14 and thechromatographic mass spectrometer subsystems 16, 18. A pump 28 may beused to transfer a fluid sample comprising the chemical agent intocontact with the analytical sampling device 14. Conduits 12, 22, 24, 26provide fluid connections between the components of the system 10.

With reference to FIG. 2, a cross-sectional view of the analyticalsampling device 14 is shown, where an outer structure 14 a having aninner surface 14 a contains an adsorbent media or material, into whichis adsorbed a focusing agent to form an embedded focusing agent region15.

The embedding of focusing agents into the thermal desorption media tubes(or other media) advances the technology of analytical fieldmeasurements. To date multiple media has been employed to capture avariety of analytes. In accordance with embodiments described herein, wefurther examined stability of the exemplary embedded focusing agents, aswell at the variability of the analytical chromatographical testingmethod, utilizing the HAPSITE® ER (INFICON GMBH). However, theembodiments and the inventive principles described herein relate tothermal desorption techniques from all manufacturers. Furthermore, theincorporation of FA may be applicable to all adsorbent media types andto other analytical techniques.

As used herein, “HAPSITE® ER” is a person-portable gaschromatograph/mass spectrometer (GC/MS) that has the capability toidentify and quantify volatile organic compounds (VOCs), TICs/TIMs,CWAs, and select semi-volatile organic compounds (SVOCs).

As used herein, “adsoprtion” is a technique that concentrates volatileand semi-volatile organic compounds onto select media, while “thermaldesorption” describes the liberation of volatile and semi-volatileorganic compounds.

As used herein, “internal standard” is a chemical compound purposelyadded to samples and/or standards at a known concentration to provide abasis for comparison in quantitation.

As used herein, “simulant” includes compounds that simulate thechemistry and reactivity of chemical warfare agents (CWAs) that haveconsiderably lower toxicity.

As used herein, “isotope dilution” means a method that improvesquantitation of known compounds by the addition of known amounts ofisotopically enriched substance(s) to the analyzed sample.

As used herein, “focusing agent” refers to compound(s) added to thermaldesorption tubes or other media to form the analytical sampling deviceof the present invention and help to correlate data acquired in theabsence of instrument calibration.

As used herein, “volatiles” refers to organic compounds that have avapor pressure of 0.01 kPa or more at 20° C.

As used herein, “semi-volatiles” refers to organic compounds that have avapor pressure less than 0.01 kPa.

As used herein, “predetermined relative retention time ratio” or“predetermined RRT ratio” refers to a ratio of the retention times (RT)for the analyte (A) with respect to the focusing agent (FA) using theanalytical chromatographical testing procedure. More specifically,RRT=RTA/RTFA.

As used herein, “relative response factor” or “RRF” refers to a ratiobetween a signal produced by the analyte on a detector associated withthe analytical chromatographic testing procedure, and the quantity ofanalyte which produces that signal, as compared to a signal produced bythe focusing agent (FA), and the quantity of the FA which produces thatsignal. More specifically, RRF is a ratio of a (Response factor ofanalyte)/(Response factor of standard).

Generally speaking, portable GC/MS (such as the HAPSITE® ER) has alimitation to compounds having a boiling point (BP) less than about 280°C.

As used herein, “LOD/LOQ” refers to “limit of detection” (LOD) and“limit of quantification” (LOQ). The LOD is generally defined as3*standard deviation (SD) of the blank, and at the LOQ is defined as10*SD of the blank. For a signal at the LOD, the alpha error(probability of false positive) is small (1%). However, the beta error(probability of a false negative) is 50% for a sample that has aconcentration at the LOD (3*SD). This means a sample could contain animpurity at the LOD, but there is a 50% chance that a measurement wouldgive a result less than the LOD. At the LOQ (10*SD), there is minimalchance of a false negative.

Embodiments of this invention incorporate compounds of known orestablished concentration onto the adsorbent media of the analyticalsampling device, such as a thermal desorption tubes comprising asorbent. While the present studies concentrated on thermal desorptionusing Tenax®-TA resin, this technique can be extended to a wide varietyof media currently employed by industry. Typical types of adsorbentmaterials include, but are not limited to, alumina, silica,carbon-containing adsorbents, a zeolite, a porous glass, a clay, aporous polymer based on 2,6-diphenyl-p-phenylene oxide, a hydrophobiccopolymer of styrene-divinylbenzene resin, a polyurethane, or acombination thereof. More specifically, adsorbent materials of principalinterest include alumina, Florisil® or other silicas, any ofcyanopropyl, diol, porous glassy carbon, or Hypersil®-ODS, all availablefrom Keystone Scientific, Bellefonte, Pa., Tenax®-TA available from EnkaResearch Institute of Holland, polyurethane foam, diatomaceous earth andzeolites. As will be appreciated by those of skill in the art, thechoice of the adsorbent material depends on the nature of the FocusingAgent (FA) and the analyte(s) (i.e., the chemical compound(s) ofinterest), as well as the ability of the analytical chromatographicaltesting procedure to liberate the adsorbed FA and analyte(s) from theadsorbent media.

For example, silica gel tubes, Anasorb® sorbent tubes, charcoal sorbenttubes, Tenax® sorbent tubes, XAD® sorbent tubes, Chromosorb® sorbenttubes, polyurethane foam (PUF) tubes, Porapak™ sorbent tubes, OSHAVersatile Sampler (OVS) sorbent tubes, or other sorbent tubes includingalumina, carbon beads, drying tubes, firebrick, florisil, glass beads,molecular sieve and soda lime tubes are known or commercially-available.

In one example, the thermal desorption tubes comprise Tenax™ TA, whichhas been stabilized for both sample volume collected and elevatedtemperatures that would be anticipated in U.S. military globalsurveillance. The adsorbent media can then be used to normalize datafrom sample to sample collected and analyzed. The experiments performedin accordance with embodiments of the present invention, showsignificant improvement in the quality of the data as measured byrelative standard deviation (RSD).

Focusing agents are chosen based on their chromatographic similarity orspectral similarity to the chemical agent(s) under investigation. Asused herein, “chromatographic similarity” is based on the selectedanalytical method for testing. For example, in one example, theanalytical testing method is GC/MS, which is therefore associated withchromatographic similarity using a gas chromatography instrument. Inanother embodiment, the analytical testing method is LC/MS, which istherefore associated with chromatographic similarity using a liquidchromatography instrument. It is preferable that the Resolution (R_(s))is sufficient to minimize interference between the signals of the FA andany analytes. R_(s)=2(t_(RB)−t_(RA))/(w_(B)+w_(A)), where:t_(RB)=retention time of solute B; t_(RA)=retention time of solute A;w_(B)=Gaussian curve width of solute B; and w_(A)=Gaussian curve widthof solute A. Chromatographically, the only critical factor is that thecompounds (i.e., the focusing agent(s) and the chemical agent(s)) eluteunder the same chromatographic analysis conditions. Therefore, for gaschromatographical subsystems, lower boiling compounds will require lowerboiling FAs and higher boiling compounds will require mid to highboiling FAs. In accordance with an embodiment of the present invention,sufficient resolution of two chromatographically similar compounds isachieved by no overlap greater than at full width half max.

As used herein, “spectral similarity” is based on the selectedanalytical method. Mass spectrometry is an analytical technique thationizes chemical species and sorts the ions based on theirmass-to-charge ratio. The mass spectrum of analytes is highly dependenton the structure of the molecule. Hence, one observes similarfragmentation from related compounds. An example is benzene, toluene andethyl benzene fragment similarly and only differ by the increase in massfrom the CH₂ groups (i.e., benzene=MW 78, toluene=92 and ethylbenzene=106). Accordingly, this attribute can be used for quantificationusing response factors (R_(f)), in that the compounds by mass spectraldetermination will provide similar R_(f)'s. Thus, in accordance with anembodiment of the present invention, a mass spectrometry subsystem isutilized for analysis, and “spectral similarity” refers to a range ofcompounds in parent molecule of M+10 and its associated fragments, asreflected in its associate fragments. Focusing agents make use of thisphenomenon and the appropriate choice should be based on compounds thatbehave similarly with regard to the fragmentation. A couple of examplesfollow:

Sarin is an organophosphorus compound with the formula(CH₃)₂CHO]CH₃P(0)F. Sarin (or “GB”) is a colorless, odorless liquid,used as a chemical weapon owing to its extreme potency as a nerve agent.It can be lethal even at very low concentrations, where death can occurwithin one to ten minutes after direct inhalation of a lethal dose, dueto suffocation from lung muscle paralysis, unless some antidotes,typically atropine and an oxime, such as pralidoxime, are quicklyadministered. It is generally considered a weapon of mass destruction.Production and stockpiling of sarin was outlawed as of April 1997 by theChemical Weapons Convention of 1993, and it is classified as a Schedule1 substance. In June 1994, the UN Special Commission on Iraqidisarmament destroyed the nerve agent sarin under Security Councilresolution 687 (1991) concerning the disposal of Iraq's weapons of massdestruction. Accordingly, sarin is an exemplary chemical agent that maybe detected and quantified in accordance with embodiments of the presentinvention.

Diisopropyl fluoro phosphate (DIFP, a non-schedule 1 compound) bears aclose relationship to sarin (GB), varying structurally by thereplacement of the methyl group in sarin with an isopropoxy group, andcan be used as an embedded standard to normalize the data to apreviously established curve on a separate instrument. This would workby creating a calibration curve of sarin and DIFB on the same instrument(i.e., instrument 1). Field measurements with instrument 2 would have ananalytical sampling device 14 embedded with a known quantity of DIFP andtesting a sample having an unknown concentration or quantity of sarin.The concentration can then be determined by correlation. Another similarcorrelation can be drawn between mustard gas (HD) and 2-chloroethylethyl sulfide (CEES).

Finally, the method can be enhanced by using isotopic analogs of thetarget compounds or the focusing agent. Since mass spectral data canseparate isotopic analogues, no previous calibration is required andeach collected tube has a calibration embedded for analysis. As anexample, diethyl malonate (MW 160) was selected as a model chemicalagent. Exemplary isotopic analogs include diethyl malonate-2-¹³C,diethyl malonate-1,2-¹³C, or diethyl malonate-1,2,3-¹³C, having MW 161,162 and 163, respectively, and whose m/z ions for quantification can be116, 117 and 118, respectively, as shown in FIGS. 3 and 4. Using thedistinct masses for the isotopic analogs can achieve a linear curve.

These in-situ embedded tubes can then be used to collect the nativediethyl malonate (MW 160) with a m/z ion at 115, as shown in FIG. 4. Inthis manner a three-point curve can be embedded yielding r² values of1.0000±0.0015 yielding RSDs of 5%, as shown in FIGS. 5 and 6. In thismanner field equipment can achieve results equal to or surpassingfix-based laboratory data. This technique can also be used as adiagnostic tool for the on-going examination of laboratory equipment.Quantitation has yielded remarkable recoveries at 100±5% (see FIG. 7).

The current work was based on the field detection and quantitation ofChemical Warfare Agents (CWA), thus, the FA were simulants that havepreviously been used in CWA work. It is our belief that the simulantswhich are semi-volatiles will have universality for other semi-volatilesthat exhibit similar chromatographic similarities. Other FAs will bechosen dependent on the chemistry and chromatography of the chemicals ofconcern. Table 1 shows exemplary CWA surrogates/simulants and anexemplary focusing agent (bromopentafluorobenzene) applicable to same.

TABLE 1 Exemplary focusing agents, simulants, and surrogates for variouschemical agents using a GC/MS technique. ION RT^(a) Focusing agentChemical Agent 117 3.37 Bromopentafluorobenzene  75 4.45 2-ChloroethylEthyl Sulfide Bis(2- chloroethyl)sulfide (HD) 127 5.15 DiisopropylFluoro Phosphate Sarin (GB) 79/97/125 5.59 Diethyl Methyl PhosphonateSarin (GB) 115 6.33 Diethyl Malonate General 120 7.35 Methyl SalicylateGeneral 226/191 8.16 Dichlorvos-d6 general 220/205 12.59 Atrazine-d5Pyrethroids ^(a)All chemicals were chromatographically identified usingthe HAPSITE ER on the CWA profile as supplied. Atrazine-d5 was used asan internal standard for the analysis of transflutherin (unpublished).The pyrethroid analyses were conducted on a Thermo-Fisher TSQ mass GCMS.The method used was the commercially available method provided byInficon for CWA analysis.

A process for incorporating focusing agents onto the adsorbent media ofthe analytical sampling device is also provided. The focusing agentswere injected as solutions in an organic solvent, such as acetonitrile,onto the thermal desorption tubes using a commercial CalibrationSolution Loading Rig (CLSR™) from Markes International, Inc, Cincinnati,Ohio at 20 psi at approximately 60 mL/min. The tubes, once loaded, werethen dry conditioned at 50° C., 50-60 mL N₂ flow, at 20 psi for 1.5hours using a Markes TC-20 (from Markes International, Inc, Cincinnati).The prepared tubes were then stored at room temperature and the analyseswere conducted over a period of one month at a minimum of three points.

Studies were performed to investigate the effect of loading temperatureon the embedding process and stability of focusing agents on theanalytical sampling device (e.g., a thermal desorption tube). Theembedding experiments were conducted at about 35° C., about 50° C.,about 65° C., about 80° C., and about 95° C. Thus, in accordance with anembodiment of the present invention, a focusing agent may be embeddedinto the analytical sampling device at a temperature in a range fromabout 35° C. to about 95° C. For example, the embedding temperature maybe about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., about 80° C.,about 85° C., about 90° C., about 95° C., or in a range between any twoof the foregoing. In another embodiment, the embedding temperature maybe in a range from about 50° C. to about 65° C., with stability to about95° C. Excellent recoveries were observed at all temperatures butsuppression was observed at an embedding temperature of about 35° C.,which may indicate that the acetonitrile used as solvent may not becompletely removed.

Initial trials were done at a constant concentration of the focusingagent. Subsequent experiments were conducted by preparing the analyticalsampling device under similar conditions, but at four concentrationlevels. Stability was measured over a period of one month. Theconditions chosen represented the highest field temperature conditionsexpected. The concentrations were chosen based on the response of thefocusing agent over a minimum of three instruments. Current experimentshave demonstrated reduction of variability as measured by relativestandard deviation (RSD) from 70% to less than 20%, where the industrystandard is generally <30% RSD.

When the procedure for adsorbing the exemplary focusing agents onto aTenax® TA resin is followed, the focusing agent has been shown to bestable for over 1 month or more (e.g., 2 months or 3 months or more) atroom temperature. Further stability testing approximated harsh samplingconditions and, again, the focusing agent on the media was stable at 60mL/min at 50° C. for 1.5 hours. The process has demonstrated theviability of the addition of a focusing agent, sampling under elevatedtemperatures. Under analytical conditions for thermal desorption, thefocusing agents were released and virtually quantitative recoveries wereobserved. Tubes prepared in this manner are expected to be used under avariety of field conditions and/or for samples returned to thelaboratory. Since it adds a known substance at a prescribedconcentration, it will also assist with determining quantities forunknown compounds that were not calibrated for.

The Focusing Agents that track sampling from field collection tolaboratory/field analysis could be applied to a variety of media packedin a multitude of configurations (tubes, dosimeters, impingers, etc.).The technique could be used across a variety of analytical applicationsand is not just limited to gas chromatography mass spectrometry. It isimmediately applicable to liquid chromatography (LC) mass spectrometryand other LC techniques with some modification. Other analyticaltechniques may also benefit, but additional work on selection of FAwould be required.

While the present invention was illustrated by the description of one ormore embodiments thereof, and while embodiments have been described inconsiderable detail, they are not intended to restrict or in any waylimit the scope of the appended claims to such detail. For example,incorporating focusing agents into other sampling devices for otheranalytical techniques is envisioned. Additional advantages andmodification will readily appear to those skilled in the art. Theinvention in its broader aspects is therefore not limited to thespecific details, representative product and method, and illustrativeexamples shown and described. Accordingly, departures may be made fromsuch details without departing from the scope of the general inventiveconcept embraced by the following claims.

What is claimed is:
 1. A system for identifying and quantifying achemical agent, the system comprising: a sample inlet; a chromatographicsubsystem fluidically coupled to the sample inlet; a mass spectrometersubsystem fluidically coupled to the chromatographic subsystem; and acalibration set comprising a plurality of analytical sampling tubeconfigured to be operably connected to the sample inlet, each analyticalsampling tube of the plurality having an adsorbent material therein anda focusing agent within the adsorbent material, each analytical samplingtube of the plurality having a different concentration of the focusingagent.
 2. The system of claim 1, wherein the adsorbent materialcomprises alumina, silica, carbon-containing adsorbents, a zeolite, aporous glass, a clay, a porous polymer based on 2,6-diphenyl-p-phenyleneoxide, a hydrophobic copolymer of styrene-divinylbenzene resin, apolyurethane, or a combination thereof.
 3. A calibration set for usewith a chromatograph and mass spectrometer, the calibration setcomprising: a plurality of analytical sampling tube configured to beoperably connected to the chromatograph and mass spectrometer, eachanalytical sampling tube of the plurality having an adsorbent materialtherein; and a focusing agent adsorbed into the adsorbent material, eachanalytical sampling tube of the plurality having a differentconcentration of the focusing agent.
 4. The calibration set of claim 3,wherein the adsorbent material comprises alumina, silica,carbon-containing adsorbents, a zeolite, a porous glass, a clay, aporous polymer based on 2,6-diphenyl-p-phenylene oxide, a hydrophobiccopolymer of styrene-divinylbenzene resin, a polyurethane, or acombination thereof.